Chemo-biological mRNA imaging with single nucleotide specificity

Knoll, Andrea; Kankowski, Svenja; Schöllkopf, Sophie; Meier, Jochen C.; Seitz, Oliver

doi:10.1039/c9cc06989e

Cited by 10 publications

(9 citation statements)

References 33 publications

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“…However, imaging of the short functional RNA pieces, including miRNAs, eRNAs, and other ncRNAs, in fixed or even in living cells still requires research on fluorogenic and hybridization-sensitive probes . Different concepts of fluorescent probes that give a light-up effect induced by hybridization to the target RNA include, for example, exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes, , DNA/RNA “traffic lights” , molecular beacons and forced intercalation thiazole orange (FIT) probes. , These fluorescent oligonucleotide probes have one thing in common that they had to be prepared using individual DNA building blocks for each of the applied fluorophores. In principle, the copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC) provides a much easier and more versatile synthetic access to fluorophore-modified oligonucleotides because any individual clickable DNA building block can be conjugated to different fluorophores.…”

Section: Introductionmentioning

confidence: 99%

Hybridization-Sensitive Fluorescent Probes for DNA and RNA by a Modular “Click” Approach

et al. 2022

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Fluorescent DNA probes were prepared in a modular approach using the “click” post-synthetic modification strategy. The new glycol-based module and DNA building block place just two carbons between the phosphodiester bridges and anchor the dye by an additional alkyne group. This creates a stereocenter in the middle of this artificial nucleoside substitute. Both enantiomers and a variety of photostable cyanine–styryl dyes as well as thiazole orange derivatives were screened as “clicked” conjugates in different surrounding DNA sequences. The combination of the (S)-configured DNA anchor and the cyanylated cyanine–styryl dye shows the highest fluorescence light-up effect of 9.2 and a brightness of approximately 11,000 M–1 cm–1. This hybridization sensitivity and fluorescence readout were further developed utilizing electron transfer and energy transfer processes. The combination of the hybridization-sensitive DNA building block with the nucleotide of 5-nitroindole as an electron acceptor and a quencher increases the light-up effect to 20 with the DNA target and to 15 with the RNA target. The fluorescence readout could significantly be enhanced to values between 50 and 360 by the use of energy transfer to a second DNA probe with commercially available dyes, like Cy3.5, Cy5, and Atto590, as energy acceptors at the 5′-end. The latter binary probes shift the fluorescent readout from the range of 500–550 nm to the range of 610–670 nm. The optical properties make these fluorescent DNA probes potentially useful for RNA imaging. Due to the strong light-up effect, they will not require washing procedures and will thus be suitable for live-cell imaging.

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Section: Introductionmentioning

confidence: 99%

Hybridization-Sensitive Fluorescent Probes for DNA and RNA by a Modular “Click” Approach

et al. 2022

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“…To eliminate residual plasmid DNA, the sample was treated with RNase free DNase (catalog no. 11119915001, Roche) as described previously (Knoll et al, 2019). Briefly, 10 units were incubated (20 min, 37 °C) with 50 μg RNA sample and purified using RNeasy Protect Mini Kit (catalog no.…”

Section: Methodsmentioning

confidence: 99%

A new triple fluorescence reporter system for discrimination of Apobec1 and Apobec3 C-to-U RNA editing activities and editing-dependent protein expression

Schweissthal

Brunken

Brach

et al. 2021

Preprint

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The human body is composed of many different cell types which communicate with each other. In particular, the brain consists of billions of neurons and non-neuronal cells which are interconnected and require tight and precise regulation of cellular processes. RNA editing is a cellular process that diversifies gene function by enzymatic deamination of cytidine or adenine. This can result in changes of protein structure and function. Altered RNA editing is becoming increasingly associated with all kind of disease, but most approaches use advanced sequencing technologies to analyze bulk material. However, it is also becoming progressively evident that changes in RNA editing have to be analyzed and considered in a cell type specific way. We present here a triple fluorescence reporter system that discriminates between Apobec1- and Apobec3-dependent C-to-U RNA editing at the single cell level. In particular, the Apobec3 reporter enables C-to-U RNA editing inducible protein expression through generation of a RNA splice donor site. We used the new system here to analyze Apobec1- and Apobec3-dependent RNA editing in primary neuron culture. The results reveal a large heterogeneity of C-to-U RNA editing in neurons and glia cells, and they show that GABAergic neurons are not able to perform Apobec1-dependent RNA editing, but Apobec3-dependent editing. Altogether, the new system can be the foundation of therapeutic application systems that counteract changes in Apobec3-dependent RNA editing in disease while simultaneously monitoring Apobec1-dependent RNA editing at the single cell level.

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“…To construct the co-expression vectors, the 2A/2A-like sequences are usually incorporated into an adenovirus [75], adeno-associated virus (AAV) [12], retrovirus [76], lentivirus [77,78], or plasmid vector [79,80]. Many other biotechnological applications that depend on the co-expression of multiple genes use 2A/2A-like sequences, e.g., the production of antibodies and antigens that can be used in vaccine production [80][81][82][83][84][85], observation of chromatin dynamics and genome (DNA and RNA) editing in the application of cell/gene therapies [78,79,[86][87][88][89][90], and development of optogenetic tools [91][92][93]. More examples of viral 2As applications can be found in [94].…”

Section: Biotechnology Applicationsmentioning

confidence: 99%

2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology

Lima

Lanza

2021

Viruses

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2A is an oligopeptide sequence that mediates a ribosome “skipping” effect and can mediate a co-translation cleavage of polyproteins. These sequences are widely distributed from insect to mammalian viruses and could act by accelerating adaptive capacity. These sequences have been used in many heterologous co-expression systems because they are versatile tools for cleaving proteins of biotechnological interest. In this work, we review and update the occurrence of 2A/2A-like sequences in different groups of viruses by screening the sequences available in the National Center for Biotechnology Information database. Interestingly, we reported the occurrence of 2A-like for the first time in 69 sequences. Among these, 62 corresponded to positive single-stranded RNA species, six to double stranded RNA viruses, and one to a negative-sense single-stranded RNA virus. The importance of these sequences for viral evolution and their potential in biotechnological applications are also discussed.

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Chemo-biological mRNA imaging with single nucleotide specificity

Abstract: The combined use of “biological” RNA imaging methods based on MS2 technology and “chemical” RNA detection by FIT probes allows unambiguous cellular imaging of a C → U edit in mRNA encoding for GlyR α2.

Cited by 10 publications

References 33 publications

Hybridization-Sensitive Fluorescent Probes for DNA and RNA by a Modular “Click” Approach

Hybridization-Sensitive Fluorescent Probes for DNA and RNA by a Modular “Click” Approach

A new triple fluorescence reporter system for discrimination of Apobec1 and Apobec3 C-to-U RNA editing activities and editing-dependent protein expression

2A and 2A-like Sequences: Distribution in Different Virus Species and Applications in Biotechnology

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